We plan to extend our previous studies on mechanisms underlying regulation of OigARs, by investigating the scaffolding protein spinophilin in aiaAR signaling/ trafficking. Preliminary data demonstrate spinophilin interacts with aiaARs witfi higher affinity than ait>ARs We also plan to extend our studies by studying matnx metalloproteases (MMPs) in mediating aiaAR signaling via EGFR transactivation compared with aibARs. Here we have a powerful new tool, a.naturally occurnng human 3* intracellular loop aia-SNP G247R, which in our preliminary data provides differential activation of specific MMP subtypes Finally, while aims 1 & 2 explore targeted, clinically relevant signaling proteins/ pathways, we recognize the importance of exploring novel binding partners Therefore our last specific aim will use Tandem-Affinity Purification combined with mass spectrometry to identify novel multi-protein complexes (the broader OiaAR signalosome) important in OiaAR signaling/ trafficking.- Unique resources and collaborators available at the Univ of WA make this approach both feasible and quantitative. AIM 1: Targeted examination ofthe scaffold protein spinophilin in aiaAR signaling and trafficking Hypothesis. Spinophilin binding to the OigAR 3^ loop mediates distinct subtype signalingArafficking 1 a Characterize spinophilin/oiaAR 3^*^ loop binding (co-immunoprecipitation assays, structure/function - analysisryeast 2-hybrid effects) & altered signaling pathways-(specific inhibitors,-siRNAs) - 1 b Examine role of spinophilin in OiaAR constitutive & agonist-induced trafficking , 1 c. Compare OiaAR sphinophilin-modulated signaling effects with OibAR and OiaAR 3* loop SNP G247R AIIM 2: Investigate role of IMMPs in EGFR transactivation in aiaAR signaling Hypothesis: Differential activation of specific MMPs by aiAR subtypes mediates distinct signalingArafTicking 2 a Characterize role of MMPs in EGFR transactivation for differential OiaAR signaling (WT vs G247SNP) 2 b Examine gene expression arrays for OigAR WT ve'rsus G247 SNP & aib to identify specific MMPs involved (confirm using specific transactivation assays and inhibitors) AIM 3' Utilize genomic/proteomic approaches to identify broader aiaAR signalosome Hypothesis. Proteomics can be used to identify novel proteins in broader multi-protein complexes (signalosome) important in unique aigAR signaling and cellular trafficking 3.a Create tandem-affinity purification (TAP)-tag constructs for OigAR, OibAR Oia-SNP, establish cell lines 3.b Use proteomics-tandem reverse-phase liquid chromatography mass spectrometry (LC-MS/MS, LTQ- FT) technology to identify multi-protein complexes important in aia versus OibAR signaling/trafficking 3 c Perturb system (agonist/antagonist drugs, G247R Oi^-SNP) to identify differential signalosomes' 3 d Compare OiaAR signalosomes in 4 cell lines commonly used in OigAR studies (rat/human with different . surface versus intracellular receptor expression to identify key proteins modulating OiaAR trafficking